Preparation derived from shark cartilage for treatment of diseases related to excessive PHF or excessive intracellular calcium

ABSTRACT

Shark cartilage extract has been shown to be an antagonist of parathyroid hypertensive factor (PHF). In view of this, shark cartilage extract can be used to treat conditions related to excessive PHF activity. Such diseases include hypertension and some other diseases related to intracellular calcium elevation. Methods for producing the shark cartilage extract and methods for administering the extract are disclosed.

FIELD OF THE INVENTION

This invention relates to an anti-parathyroid hypertensive factor(anti-PHF) derived from shark cartilage. The compounds of the presentinvention can be used in the treatment of hypertension, and otherdiseases related to intracellular calcium elevation (e.g., non-insulindependent diabetes mellitus; atherosclerosis; congestive heart failure;cancer (including breast, colon, kidney and leukemia); inflammatorybowel disease and asthma.

BACKGROUND OF THE INVENTION

Hypertension is generally defined as the elevation of the systolicand/or diastolic arterial blood pressure above a nominal value of 140/90mm Hg. Diseases associated with hypertension include artherosclerosis,hypertensive renal failure, stroke, congestive heart failure andmyocardial infarction. Although numerous methods of treatment have beenfound to be effective in the reduction of arterial blood pressure, theetiology of essential hypertension remains essentially unknown. Agenetic predisposition to hypertension is generally accepted, but thenumber of different drugs which have been found effective in thetreatment of hypertension, and the fact that these drugs seem to operateby eliciting different pharmacological responses, suggests that theremay be different primary causes for essential hypertension.

A number of studies have suggested that one or more circulating factorsmay play a role in the genesis or the maintenance of hypertension [See:Wright et al., A Hypertensive Substance Found in the Blood ofSpontaneously Hypertensive Rats; Life Sci. 1984; 34:1521-1528; Dahl etal., Humoral transmission of Hypertension: Evidence from Parabiosis;Circ. Res. 1969; 24/25 (Supp. I): 21-23; Greenberg et al., Evidence forCirculating Factors as a Cause of Venous Hypertrophy in SpontaneouslyHypertensive Rats; Am. J. Physiol. 1981; 241:H421-H430; Tobian et al., ACirculating Humoral Pressor Agent in Dahl S Rats with Salt Hypertension;Clin. Sci. 1979; 57:345s-347s; Zidek et al., Humoral Factors in thePathogenesis of Primary Hypertension: Klin. Wochenschr. 1985; 63 (Supp.II) D:94-96; Hirata et al., Hypertension Producing Factor in the Serumof Hypertensive Dahl Salt-Sensitive Rats; Hypertension 1984; 6:709-716].For example, in parabiosis and cross-circulation experiments, anincrease in blood pressure could be induced in normotensive animals byexposure to blood from hypertensive animals. The subcutaneous injectionof erythrocyte-associated factor obtained from spontaneouslyhypertensive rates (SHR) has been shown to induce hypertension innormotensive Wistar-Kyoto (WKY) rats and an increase in blood pressurecan be induced in normotensive, salt insensitive Dahl rats by injectionof serum from hypertensive, salt-sensitive Dahl rats.

There have also been reports of circulating factors in both hypertensiverats and hypertensive humans which increase intracellular calcium [See:Banos et al., Two Factors Associated with Increased Uptake of Calcium inPlatelets from Essential Hypertensive Patients; Clin. Exp. Hypertens.1987; 9:1515-1530; Zidek et al., Effect of Plasma from HypertensiveSubjects on Ca Transport in Permeabilized Human Neutrophils; Clin. Sci.1988; 74:53-56; Linder et al., Effects of a Circulating Factor inPatients with Essential Hypertension on Intracellular Free Calcium inNormal Platelets; N. Eng. J. Med. 1987; 316:509-513; Bruschi et al.,Cytoplasmic Free Ca is Increased in the Platelets of SpontaneouslyHypertensive Rats and Essential Hypertensive Patients; Clin. Sci. 1985;68:179-184; Wright et al., Stimulation of Aortic Tissue Calcium Uptakeby an Extract of Spontaneously Hypertensive Rat erythrocytes PossessingHypertensive Properties; Can. J. Physiol. Pharmacol. 1986;64:1515-1520]. Since vascular tone is influenced by the level ofintracellular calcium, factors which increase blood pressure and factorswhich increase intracellular calcium may be related. There has beenaccumulating evidence suggesting the involvement of calcium regulatinghormones in some forms of hypertension [See: L. M. Resnick, Am. J. Med.82 (Supp. 1B), 16 (1987)]. Parathyroid hormone (PTH) is a calciumregulating hormone. Thirty percent or more of essential hypertensivepatients fall into a subgroup characterized by increased levels ofimmunoreactive parathyroid hormone (ir-PTH). [See: Laragh et al., KidneyInt. 34, (Supp. 35), S162 (1988)]. An increase in PTH levels has beenreported in SHR rats [See: McCarron et al., Hypertension 3 (Supp. 1),I162 (1981)] and it has been observed that hyperparathyroid patientsoften exhibit hypertension, the severity of which can, in most cases, bereduced by parathyroidectomy [See: Hellstrom et al., Brit. J. Urol. 30,13 (1958)]. Similar results from parathyroidectomy have also beenreported in SHR rats. [See: Schleiffer et al., Jap. Circ. J. 45, 1272(1981)]. Various investigators have suggested that PTH contributes tothe development of essential hypertension, although exogenousadministration of PTH causes a reduction in blood pressure in mammalsand other vertebrates [See: Pang et al., Gen. Comp. Endocrinol. 41, 135(1980)]. The vasodilating action of PTH is also related to a specificregion of the molecule separate from the region mediating hypercalcemiceffects [See: Pang et al., Endocrinology, 112, 284 (1983)]. PTH has alsobeen shown to inhibit calcium entry into vascular smooth muscle [See:Pang et al., Life Sci., 42, 1395 (1988)] through L-type calcium channels[Wang et al. FEBS, Vol. 282, No. 2, pp. 331-334 (1991)]. This paradox isfurther heightened by the fact that hypertensive patients with increasedPTH levels exhibit decreased serum ionized calcium levels [See: Resnicket al., New Engl. J. Med., 309, 888 (1983); Hvarfner et al., Acta Med.Scand. 219, 461 (1986)]. It would be expected that the serum ionizedcalcium levels would be elevated if PTH were primarily elevated.

The existence of a circulating factor in the blood of the SHR rat wasconfirmed by the studies reported in Am. J. Hypertens., 2, 26-31 (1989).In these studies, an increase in the blood pressure of WKY and SD ratswhen plasma from SHR rats was injected into the normotensive rats eitherby infusion or by bolus injection was shown. In addition, it has beenshown that the uptake of ⁴⁵Ca by sections of the tail artery of a rat,in vitro, increased in a dose-dependent manner as the concentration ofSHR plasma was increased in a buffer-based medium. The results of theseexperiments clearly show that an increase in blood pressure and anincrease in calcium uptake in the cells were both dose-dependent on theamount of SHR plasma present and available in the system. Curiously, theonset of both events was delayed, and gradual, whereas known endogenouspressor agents such as norepinephrine, angiotensin II and vasopressinhave been observed to increase blood pressure quite rapidly afteradministration. The known endogenous pressor agents exhibit about a 1-2minute onset in the increase of blood pressure and increase in calciumuptake in the cells whereas parathyroid hypertensive factor has a 20-30minute delay before such onset. Another result observed in these studieswas that when the infusion of SHR plasma was stopped and substitutedwith plasma from normotensive rats, the observed blood pressuredecreased quite rapidly to the baseline. The decrease observed precludeda simple volume effect. In a related experiment, dialyzed plasma fromhypertensive human subjects was infused into normotensive SD rats andshown to produce hypertension. Plasma from these subjects also increasedcalcium uptake in rat tail arteries in vitro. Dialyzed plasma fromnormotensive patients produced no significant increase in bloodpressure.

The origin of the circulating factor was unknown, but the anecdotalreports that PTH was elevated in hypertensive rats suggested theparathyroid gland as a target of investigation. Parathyroidectomies ofSHR rats were found to reduce blood pressure and plasma from the SHRrats which had been parathyroidectomized did not cause elevation ofblood pressure in normotensive rats. Conversely, transplantation ofparathyroid glands from SHR rats to normotensive Sprague-Dawley (SD)rats resulted in an increase in blood pressure and the appearance of thefactor in the plasma, as shown by infusion of the isolated plasma intoother normotensive rats. [Pang and Lewanczuk, Amer. J. Hypertens. 2, 898(1989)].

On the basis of these studies, the parathyroid was determined to be theorigin of the circulating factor and the name “Parathyroid HypertensiveFactor” or PHF was proposed for the substance which causes an elevationin blood pressure.

The isolation and purification of a circulating factor, having itsorigin in the parathyroid gland, has been demonstrated in SHR rats andin many humans having essential hypertension and is the subject matterof related patent application Ser. No. 603,745 filed Nov. 21, 1990,which is a continuation-in-part of patent application Ser. No. 327,450,filed Mar. 22, 1989, now abandoned. The disclosure of the related patentapplications are incorporated herein by reference for their teachings,including the teachings of purification of parathyroid hypertensivefactor.

As described in the aforementioned related patent applications, PHF hasbeen shown to regulate extracellular calcium uptake, and can beinhibited by increases in dietary calcium levels. PHF has been isolatedand a method for screening for PHF using antibodies raised against PHFhave been described. PHF has a molecular weight of approximately 2,700daltons and has the property of delayed onset of an increase in bloodpressure of a normotensive rat when administered thereto, the increasein blood pressure temporally correlating with an increase inextracellular calcium uptake by vascular smooth muscle. From bioassaydata, the factor in humans and rats has been found to be substantiallysimilar.

Vascular hypertrophy has been implicated in the pathophysiology of anumber of cardiovascular diseases including essential hypertension.Vascular smooth muscle proliferation could account for vascularhypertrophy and increased vascular tone. It was reported that PHFincreased vascular smooth muscle cell proliferation through a mechanismindependent of intracellular calcium regulation (Shan et al., Abstractin 17th Scientific Meetings of the International Society ofHypertension, Amsterdam, 7-11 Jun. 1998).

Antagonists of PHF have been found by the present inventors. The presentinventors have unexpectedly found that shark cartilage, known in the artto contain a substance which inhibits tumor angiogenesis [Lee et al.,Science, vol. 221, pp. 1185-1187, (1983)] and to contain ananti-inflammatory component [Schinitsky U.S. Pat. No. 4,473,551], actsas an antagonist of PHF resulting in a decrease in blood pressure andaffecting intracellular calcium regulation. The present inventors havealso found that shark cartilage extract inhibited VSMC proliferation inSHR rats or in WKY rats induced by PHF. In view of this, shark cartilageextract according to the present invention is expected to be useful fortreating hypertension and other diseases related to intracellularcalcium elevation.

DETAILED DESCRIPTION OF THE INVENTION

The present inventors have found that an extract prepared from sharkcartilage produces a decrease in blood pressure. The shark cartilageextract is believed to contain a parathyroid hypertensive factorantagonist which binds to the parathyroid hypertensive factor sitewithout activating parathyroid hypertensive factor activity.

The shark cartilage extract can be obtained by further purifyingcommercially available shark cartilage which has been cleaned, dried andmilled to a fine powder. The dried ground shark cartilage is firstextracted with H₂O at a temperature between 4-120° C. (preferably 95°C.) for 2-4 hours (preferably 2 hours). The ratio of solute to solventis between 1:8 and 1:12. The resulting suspension is then cooled tobetween 40-60° C. (preferably 50° C.) and centrifuged at about 5200 to5700 rpm to separate the suspension into a supernatant (#1) and pellet.The supernatant (#1), which contains about 8% solids, is held in acooling tank at 4-8° C. while the pellet is subjected to a secondextraction. In the second extraction the pellet is extracted with H₂O ata temperature between 4-120° C. (preferably 95° C.) for 2-4 hours(preferably 2 hours). The ratio of solute to solvent is 1:4-1:6 (basedon starting material). The resulting suspension is then cooled tobetween 40-60° C. (preferably 50° C.) and centrifuged at about 5200 to5700 rpm to separate the suspension into a supernatant and pellet. Thesupernatant is then pooled with the supernatant from the firstextraction and spray dried to obtain the purified shark cartilageextract of the present invention.

The extract of the present invention may be administered to a warmblooded mammal in need of such treatment, by parenteral, topical, oralor rectal administration or by inhalation. The extract may be formulatedfor parenteral or oral dosage by compounding the extract with aconventional vehicle, excipient, binder, preservative, stabilizer,color, agent or the like as called for by accepted pharmaceuticalpractice.

For parenteral administration, a 1-10 ml intravenous, intramuscular orsubcutaneous injection would be given one to four times daily. Theinjection would contain the shark cartilage extract of the presentinvention in an aqueous isotonic sterile solution or suspensionoptionally with a preservative such as phenol or a solubilizing agentsuch a ethylenediaminetetraacetic acid (EDTA). Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. Syntheticmonoglycerides, diglycerides, fatty acids (such as oleic acid) find useas fixed oils in the preparation of injectables.

For rectal administration, the extract can be prepared in the form ofsuppositories by mixing with a suitable non-irritating excipient such ascocoa butter or polyethylene glycols.

For topical use, the extract can be prepared in the form of ointments,jellies, solutions, suspensions or dermal adhesive patches.

In a powdered aerosol, the extract may be administered by a Spinhalerturbo-inhaler device obtained from Fisons Corporation of Bedford,Massachusetts, at a rate of about 0.1 to 50 mg per capsule, 1 to 8capsules being administered daily for the average human. In a liquidaerosol, the extract is administered at the rate of about 100 to 1000micrograms per “puff” or activated release of a standard volume ofpropellant. The liquid aerosol would be given at the rate of 1 to 8“puffs” per day with variation in dosages due to the severity of theconditions being treated, the weight of the patient and the particlesize distribution of the aerosol. A fluorinated hydrocarbon or isobutanecan be used as propellants for liquid aerosols.

Daily doses are in the range of about 0.01 to about 200 mg per kg ofbody weight (preferably 1-10 mg/kg body weight) depending on theactivity of the specific compound, the age, weight, sex and conditionsof the subject to be treated, the type and severity of the disease, thefrequency and route of administration. As would be well known, theamount of active ingredient that may be combined with the carriermaterials to produce a single dosage will vary depending upon the hosttreated and the particular mode of administration.

The shark cartilage extract can also be combined with drugs known to beeffective for treating the condition in question. For example, to treathypertension, shark cartilage extract can be combined with knownantihypertensive drugs such as calcium channel blockers (e.g. verapamil,nifedipine and diltiazem).

In addition to the treatment of essential hypertension, the extract ofthe present invention can be used to treat other diseases which mayinclude but do not necessarily include hypertension as a primarysymptom. For example, noninsulin dependent diabetics are frequentlyhypertensive. Conversely, hypertensives frequently show an impairedglucose tolerance. Thus, shark cartilage extract is expected to beuseful for treating hypertension and other diseases related tointracellular calcium elevation.

The present invention is intended to encompass the isolation,identification and synthetic production of the active ingredient fromshark cartilage extract.

The following examples illustrate but are not intended to limit thepresent invention. Various modifications may be apparent to thoseskilled in the art without deviating from the scope of this invention.

EXAMPLE 1 Extraction of Shark Cartilage

Cleaned, dried, ground shark cartilage was purchased. The dried groundshark cartilage was first extracted with H₂O at a temperature between85° to 90° C. for 2 hours. The ratio of solute to solvent was 1:8. Theresulting suspension was then cooled to 50° C. and centrifuged at 5200rpm (3245 g) to separate the suspension into a supernatant and pellet.The supernatant, which contained about 8% solids, was held in a coolingtank at 4° C. while the pellet was subjected to a second extraction. Inthe second extraction the pellet was extracted with H₂O at 95° C. for 3hours. The ratio of solute to solvent was 1:4.8 based on the startingmaterial. The resulting suspension was then cooled to 50° C. andcentrifuged at 5200 rpm (3245×g) to separate the suspension into asupernatant and pellet. The supernatant, which contained about 3%solids, was pooled with the supernatant from the first extraction andspray dried to obtain the purified shark cartilage extract of thepresent invention.

EXAMPLE 2 Effect of Bolus Injection of Shark Cartilage Extract (1 mg/kg)in SHR and SD Rats

Six (6) spontaneously hypertensive rats (SHR) and three (3)Sprague-Dawley (SD) rats were given an intravenous bolus injection ofshark cartilage extract denoted as DFI-40. Five (5) spontaneouslyhypertensive rats (SHR) and three (3) Sprague-Dawley (SD) rats weregiven an intravenous bolus injection of shark cartilage extract denotedas DF II-40. The shark cartilage extract was administered at a dosage of40 mg/kg body weight. Blood pressure was measured for 90 minutes afterthe injection. As shown in FIG. 1, the shark cartilage extract producedno effect in SD rats but decreased the blood pressure in SHR rats.

EXAMPLE 3 Effect of Gavage Administration of Shark Cartilage Extract onSHR and SD Rats

Three groups of SHR rats were gavage fed with three different doses ofshark cartilage extract (10, 20 and 40 mg/kg) from batch DF II-53. 11rats were administered 10 mg/kg body weight shark cartilage extract, 4rats were administered 20 mg/kg body weight shark cartilage extract and4 rats were administered 40 mg/kg body weight shark cartilage extract.Blood pressure was measured for 90 minutes after administration. Asshown in FIGS. 2, 2 a and 2 b, all of the rats showed a decrease inblood pressure which was dose related. In rats given higher doses,(20-40 mg/kg body weight), the rate of decrease in blood pressure isgreater with the maximum decrease being reached at around 50-60 minutes(FIG. 2 a). After 50-60 minutes, the blood pressure fluctuates possiblydue to the blood pressure regulating mechanisms of the rat.

EXAMPLE 4 Effect of PHF on the Blood Pressure of SD Rats in the Presenceand Absence of Shark Cartilage Extract

Seven (7) SD rats were administered 1 ml equivalent of PHF by IV bolusinjection. Six (6) SD rats were administered 1 ml equivalent of PHF byIV bolus injection and 10 minutes later 40 mg/kg body weight of sharkcartilage extract (DF II-53) was administered. Blood pressure wasmeasured for 90 minutes following the injections. As shown in FIG. 3,PHF produces a delayed increase in blood pressure and the sharkcartilage extract counteracts this response.

EXAMPLE 5 Effect of PHF on Vascular Smooth Muscle Cell (VSMC)Proliferation in the Presence and Absence of Shark Cartilage Extract

The tail artery of male West Kyoto (WKY) rats or SpontaneousHypertensive Rats (SHR) (100-200 g body weight) was dissected out andimmersed in the cold Ca-omitted and Mg-omitted Hanks' balanced saltsolution (HBSS) (Gibco, Grand Island, N.Y.). The tail artery wasdigested twice with HBSS enzyme solution II and I consecutively. Eachdigestion lasted for 1 hour. HBSS enzyme solution I contained CaCl2 (0.2mM), collagenase/dispase (1.5 mg/ml) (Boehringer Mannheim GmbH, WestGermany), elastase (Type I, 0.5 mg/ml) (Sigma Chemical Co., St. Louis,Mo.), trypsin inhibitor (Type I, 1 mg/ml) (Sigma Chemical Co.) andbovine serum albumin (BSA) (fatty acid free, 2 mg/ml) (Sigma ChemicalCo.). HBSS enzyme solution II contained collagenase (Type II, 1 mg/ml)(Sigma Chemical Co.), trypsin inhibitor (0.3 mg/ml) and BSA (2 mg/ml).The cell suspension were then seeded into 96 flat-bottom well tissueculture plates in DMEM medium with 10% FCS and incubated at 37° C. in ahumidified atmosphere with 5% CO2 in air for 36 hours to allow cellsattachment to the bottom of the plate. The medium was changed to DMEMwith 0.4% of FCS to render the cells quiescent for 2-4 days. Thisprocedure synchronised cells in the Go-G1 boundary. PHF and sharkcartilage were dissolved in DMEM with 10% FCS. PHF alone or PHF plusshark cartilage was added into the quiescent cells. After incubation for36 hours, the cells were pulsed with 3H-thymidine (0.2 (/well andincubated for another 24 hours. The medium was then removed and thecells were washed twice with HBSS followed by a 15-30 minutes incubationwith 0.1% of trypsin at room temperature. The cells were then harvestedonto filter paper by the cell harvest. The amount of radioactivityincorporated into cells was determined using a liquid scintillationcounter. As shown in FIG. 4, PHF stimulated VSMC cell proliferation inWKY rats. FIG. 5 shows that the stimulating effect of PHF on VSMC in WKYrats can be inhibited by shark cartilage extract. FIG. 6 shows thatshark cartilage inhibited VSMC proliferation of SHR rats.

EXAMPLE 6 Chemical Composition of Shark Cartilage Extract

(1). Determination of Protein Content

Total protein content is determined using the BCA method. The BCAProtein Assay Reagent is purchased from the PIRRCE. A standard curve ofprotein standards of known concentration can be constructed by using theBSA (bovine serum albumin) standard solution provided with the BCAProtein Assay Reagent Kit. Twenty-four glass tubes were set in threerows and seven columns for standard samples and another four tubes wereset for spectrophotometer calibration. Ninety-five, 90, 80, 70, 60, 50,40, and 30 μl of 0.9% sodium chloride was applied into the first row ofthe tubes respectively. The same procedure was repeated for the secondand third rows. Five, 10, 20, 30, 40, 50, 60, and 70 μl of standardprotein (provided with the kit and at a concentration of 2 mg/ml) wereapplied into the first row of tubes containing 0.9% sodium chloride. Thesame procedure was repeated for the second and third rows. Two mls ofthe Working Reagent, which is a mixture of 50 parts of Reagent A and 1part of Reagent B was then added to each tube. All samples were wellmixed and incubated at 37° C. for 30 min. Protein was determined bymeasuring the absorbency at 562 nm with a spectrophotometer (Model PU8620 UVNIS/NIR, Philips). The mean values of each concentration ofstandards were calculated and a standard curve was constructed by usingthe Analysis of the Regression Line No. 5, Pharmcologic CalculationSystem-Version 4.2A. This standard curve was used to determine theprotein concentration for each unknown sample. 1% of shark cartilageextract solution was prepared in double distilled (DD) water. Theprotein concentration (mg/ml) of the sample solution was calculated byusing the standard curve and shark cartilage protein content bypercentage was calculated by using the following formulation:

Protein %(w/w)=sample protein concentration (mg/ml)×dilution factor(2.5)/sample concentration (10 mg/ml)×100.

To obtain accurate data for the standard curve and shark cartilagesample, the procedure for standard curve construction and sharkcartilage extract protein content determination were carried outsimultaneously, the Working Reagent was the last reagent added into alltubes for the standard protein samples and the shark cartilage sample.

The protein content is 15.11 (2.79 (%) in a total of 16 batches of sharkcartilage extract.

(2). Determination of Mucopolysacchrides

The method was adapted from P. Whiteman (Biochem. J. 131:351-357, 1973)and E. Gold (Analytical Biochemistry 99: 183-188, 1979). Standard sampleChondroitin Sulfate C was purchased from Sigma chemical Co., Cat No.C-4384, Lot No. 21H0103. Standard or samples were prepared by dissolving10 mg Chondroitin Sulfate C or shark cartilage extract in 50 ml DDwater. Reaction reagent was prepared by dissolving 20 mg Aleian Blue 8GXin 20 ml buffer (5.07 g magnesium chloride and 3.4 g sodium acetate in500 ml water) and 0.2 ml acetic acid. A series of shark cartilageextract samples ranging 40-200 μg in 1 ml was added into a 50 ml-plastictube respectively. One ml of reaction reagent was added into thesetubes. The mixture was equilibrated for 2 hours at room temperature withstirring. Twenty ml of 95% ethanol was added followed by centrifugation.After decanting the supernatant, three ml of 0.2M calcium chloride wasadded to the precipitate. The mucopolysaccharide content was determinedby measuring the absorbency of the calcium chloride solution ofprecipitate at 620 nm.

The mucopolysaccharide content was 50.33 (2.25 (%) in 6 batches of sharkcartilage extract.

(3). Isolation and Determination of Chondroitin C

The method was adapted from L. Roden, et al., Methods in Enzymology(1972), Vol. 28, Complex Carbohydrates part B, ed. by V. Ginsburg.Amberlite IR-120 Plus was purchased from Sigma Chemical Co., Cat No.IR-120 Plus. Calcium acetate buffer was prepared by adding 1.2L DD waterto 62.5 g calcium acetate. pH was adjusted to 4.5 with 35.5 ml glacialacetic acid. Two grams of shark cartilage extract was added to 400 ml ofcalcium acetate buffer in a 2L-glass flask. Sample solution was heatedin a water bath at 37° C. for 20 min, then cooled to room temperature.Ethanol (100%, 116.25 ml) was added to the sample solution very slowlywith vigorous stirring at room temperature. Set the flask at 4° C. bathfor 3 hours followed by centrifugation (11,000 rpm, 19,000 g) for 15 minat 4° C. The precipitate was dissolved in DD water and freeze dried. Thesupernatant was warmed to room temperature and was added into 80 ml ofethanol (100%) slowly with vigorous stirring. The flask was set in 4° C.bath again overnight with slow stirring. The solution was centrifuged at4° C. (11,000 rpm) for 15 min. The second precipitate was dissolved inDD water and freeze dried, The supernatant was warmed to roomtemperature and 100 ml ethanol was added slowly with vigorous stirring.Again the flask was set in 4° C. bath overnight with slow stirringfollowed by centrifugation at 11,000 rpm for 15 min. DD water (125 ml)was added to the third precipitate which was applied to an AmberliteIR-120+(Na+ form) column (2.5×16 cm, about 60 g of Amberlite IR-120Plus). The column was washed with 75 ml of DD water. After adding 1.168g NaCl to make the solution 0.1M in salt 3 volumes (600 ml) of absoluteethanol was applied with vigorous stirring. Again, the flask was placedin 4° C. bath overnight followed by centrifugation (11,000 rpm) at 4° C.for 15 min. The last precipitate was dissolved in DD water and freezesdried. The weight of last precipitate represents the amount ofchondroitin sulfate C.

The chondroitin sulfate C content was 5.9 (1.98 (%) in 2 batches ofshark cartilage extract.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of an IV bolus injection of shark cartilageextract in SHR and SD rats. As shown in FIG. 1, the shark cartilageextract produced no effect in SD rats but decreased the blood pressurein SHR rats.

FIG. 2 shows the results of gavage administration of shark cartilageextract in SHR rats. As shown in FIG. 2, the shark cartilage extractproduced a decrease in blood pressure in all of the rats.

FIGS. 2 a and 2 b show that the decrease in blood pressure is doserelated and the maximum decrease is reached at around 50-60 minutes.

FIG. 3 shows that PHF produces a delayed increase in blood pressure andthe shark cartilage extract counteracts this response.

FIG. 4 demonstrates that PHF stimulated VSMC of WKY rats proliferationin a dose-dependent manner. At the doses of 0.625×10−3, 1.25×10−3 and2.5×10−3 absorption unit, PHF increased cell proliferation by 120(8.5(%) (P<0.05, n=16), 137.91 (12(%) (P<0.01, n=16) and 181.9 (14.3 (%)(P<0.05, n=16) respectively.

FIG. 5 shows the effect of PHF on VSMC of WKY rats proliferation in thepresence of shark cartilage extract. At dose of 50 (g/ml, sharkcartilage extract significantly inhibits VSMC proliferation induced byPHF.

FIG. 6 shows the effects of shark cartilage extract on VSMC of SHR rats.At the doses of 5, 50 and 500 (g/ml, shark cartilage extract inhibitsVSMC proliferation in a dose-dependent manner.

1. A method for treating a disease related to excessive PHF comprising:administering to a patient in need of such treatment, an amount of sharkcartilage extract with anti-PHF activity effective to treat saiddisease, wherein the shark cartilage extract is produced by thefollowing steps: extracting cleaned, dried, ground shark cartilage withH₂O at a temperature between 85-120° C. for 2-4 hours, centrifuging theresulting suspension 1 at between 5200 to 5700 rmp to separate thesuspension into supernatant 1 and pellet, holding the supernatant 1 in acooling tank 4-8° C., extracting the pellet a second time with H₂ 0 at atemperature between 95-120° C. for 2-4 hours, centrifuging the resultingsuspension 2 at between 5200-5700 rpm to separate the suspension intosupernatant 2 and pellet; pooling supernatant 1 with supernatant 2, andspray drying the pooled supernatants to obtain the shark cartilageextract.
 2. A method for counteracting the activity of parathyroidhypertensive factor, comprising administering an effective amount ofshark cartilage extract with anti-parathyroid hypertensive factor,wherein the shark cartilage extract is produced by the following steps:extracting cleaned, dried, ground shark cartilage with H₂ 0 at atemperature between 85-120° C. for 2-4 hours, centrifuging the resultingsuspension 1 at between 5200 to 5700 rmp to separate the suspension intosupernatant 1 and pellet, holding the supernatant 1 in a cooling tank4-8° C., extracting the pellet a second time with H₂ 0 at a temperaturebetween 95-120° C. for 2-4 hours, centrifuging the resulting suspension2 at between 5200-5700 rpm to separate the suspension into supernatant 2and pellet; pooling supernatant 1 with supernatant 2, and spray dryingthe pooled supernatants to obtain the shark cartilage extract.